The present invention relates to methods of identifying antibacterial agents and more particularly, to novel antibacterial agents which are capable of preventing or disrupting binding between antitoxin and toxin polypeptides of bacterial cells.
Presently, treatment of infections caused by pathogenic bacteria relies predominantly on the administration of antibiotics. Antibiotics currently being used against bacterial pathogens include β-lactams (e.g., penicillin and cephalosporin) and glycopeptides (e.g., vancomycin and teichoplanin), which act to inhibit the final step in peptidoglycan synthesis, quinolones, which inhibit bacterial DNA replication, inhibitors of bacterial RNA polymerase such as rifampin, and aminoglycosides (e.g., kanamycin and gentamycin). Other well-known antibiotics include inhibitors of enzymes participating in production of tetrahydrofolate (e.g., sulfonamides).
Despite being successful in controlling or eliminating bacterial infections, widespread use of antibiotics both in human medicine and as a feed supplement in poultry and livestock production has led to drug resistance in many pathogenic bacteria (McCormick J. B., Curr Opin Microbiol 1:125-129, 1998) and as such, the effectiveness of such antibiotics has greatly diminished in the last decade.
The rapid and widespread development of resistance in pathogenic bacteria is illustrated by the fact that presently almost half of the clinical strains of Haemophilus ducreyi, the causative agent of chancroid, carry genes which confer resistance to amoxicillin, ampicillin and a series of other β-lactams (Prachayasittikul et al., Southeast Asian J Trop Med Public Health 31:80-84, 2000). Likewise, the incidence of resistance towards tetracyclines among clinical strains of Salmonella typhimurium has increased from zero in 1948 to 98% by 1998 (Teuber M., Cell Mol Life Sci 30:755-763, 1999).
The economic impact of managing infections caused by antibiotic-resistant bacteria is substantial, and current costs are estimated to be more than $4 billion annually [Harrison and Lederberg (ed.), Antimicrobial resistance: issues and options. National Academy Press, Washington, D.C. pp. 1-7, 1998]. Furthermore, as resistance spreads among bacteria, there is grave concern that antibiotics treatment will become increasingly less effective and, in some cases, completely ineffective.
This rapidly increasing appearance of bacterial resistance to antibiotics has driven researchers to search for new agents that possess activity against antibacterial drug-resistant strains. Although several approaches can be utilized to achieve this goal, the most generalized would be the discovery and clinical development of an agent that acts on a new target which has not yet experienced selective pressure in the clinical setting. Such a target should be essential to the growth and survival of bacteria and be sufficiently different from similar macromolecules present in the human host (Goldman and Gange, Curr Med Chem 7:801-820, 2000).
The Toxin-antitoxin complex of bacteria includes a pair of polypeptides that is encoded by bacterial plasmids and chromosomes. It is postulated that in bacteria these polypeptides function to induce programmed cell death or growth inhibition in response to starvation or other adverse conditions (Hayes, Science 301:1496-1499, 2003). The antitoxins neutralize the cognate toxins by forming tight complexes therewith. The antitoxins are unstable due to degradation by cellular proteases (e.g., Lon or Clp), whereas toxins are stable polypeptides. Toxin-antitoxin pair examples include the pemI-pemK genes of plasmid R100, the phd-doc genes of phage P1, and the ccdA-ccdB genes, of plasmid F (Couturier et al., Trends Microbiol. 6:269-275, 1998; Engelberg-Kulka and Glaser, Annu. Rev. Microbiol 53:43-70, 1999; Jensen and K Gerdes, Mol. Microbiol. 17:205-210, 1995). Toxin-antitoxin pairs are thought to increase the stability of extrachromosomal elements by selectively killing plasmid-free cells, resulting in the proliferation of plasmid-harboring cells in the population (Holcík and Iyer, Microbiology 143:3403-3416, 1997; and Grady and Hayes, Mol. Microbiol. 47:1491-1432,2003). Several toxin-antitoxin encoding gene analogues have been identified on the E. coli K-12 chromosome, such as mazE-mazF (also known as chpAI-chpAK), sof-gef, kicA-kicB, relB-relE, chpBI-chpBK and yefM-yoeB (Grady and Hayes, Mol. Microbiol. 47:1491-1432, 2003; Aizenman et al., Proc. Natl. Acad. Sci. USA 93:6059-6063, 1996; Feng et al., Mol. Gen. Genet. 243:136-147, 1994; Gotfredsen and Gerdes, Mol. Microbiol. 29:1065-1076, 1998; Masuda et al., J. Bacteriol. 175:6850-6856, 1993, and Poulsen et al., Mol. Microbiol. 3:1463-1472, 1989).
Although the use of toxin encoding polynucleotides for inducing bacterial cell death has been recently suggested (Westwater et al., Antimicrobial Agents and Chemotherapy 47: 1301-1307, 2003), the prior art does not teach or suggest prevention or disruption of toxin-antitoxin binding for the purpose of inducing death in bacterial cells.
While reducing the present invention to practice, the present inventors have identified the site of interaction between bacterial toxin and antitoxin polypeptides thus enabling for the first time to identify or design novel antibiotics which target this site of interaction and thus enable bacterial cell killing.